Horses (Equus Caballus) are among the domesticated animals valued by breeders and enthusiasts for their variety and beauty of coat color and patterns. The genetic mechanisms involved in several different variations of coloration and patterning in horses have been reported including; chestnut, frame overo, cream, black, silver dapple, sabino-1 spotting, tobiano spotting and dominant white spotting (Marklund et al. 1996; Metallinos et al. 1998; Mariat et al. 2003; Reider et al. 2003; Brunberg et al. 2006; Brooks and Bailey 2005; Brooks et al., 2007; Haase et al. 2007). Although there are several inherited ocular diseases reported in the horse (cataracts, glaucoma, anterior segment dysgenesis, and congenital stationary night blindness), the causative genetic mutations and the pathogenesis of some of these ocular disorders remain unknown.
Appaloosa spotting is characterized by patches of white in the coat that tend to be symmetrical and centered over the hips. In addition to the patterning in the coat, appaloosa spotted horses have three additional pigmentation traits; striped hooves, readily visible nonpigmented sclera around the eye, and mottled pigmentation around the anus, genitalia, and muzzle (Sponenberg and Beaver 1983). The extent of spotting varies widely among individuals, resulting in a collection of patterns which are termed the “leopard complex” (Sponenberg et al. 1990). This variation encompasses a broad spectrum of patterns; including those possessing very minimal patches on the rump (known as a “lace blanket”), a white body with many oval or round pigmented spots dispersed throughout (known as “leopard”, from which the genetic locus is named), as well as a nearly complete depigmentation (known as “fewspot”) (FIG. 1). A single autosomal dominant gene, Leopard Complex (LP), is thought to be responsible for the inheritance of these patterns and associated traits, while modifier genes are thought to play a role in determining the amount of white patterning that is inherited (Miller 1965; Sponenberg et al. 1990; Sponenberg et al. 2009). Horses that are homozygous for appaloosa spotting (LP/LP) tend to have fewer spots of pigment in the white patterned areas; these horses are known as “fewspots” (largely white body with little to no spots) and “snowcaps” (white over the croup and hips with little to no spots) (Sponenberg et al. 1990; Lapp & Carr 1998). Leopard complex spotting is characterized as a group of white spotting patterns that occurs in several breeds of horses including, among others, Appaloosas, Knabstruppers, Norikers, Australian spotted ponies, British spotted ponies, Pony of the Americas, and American Miniature horses.
The spotting pattern (aka appaloosa), caused by an incompletely dominant gene (LP), is a highly valued trait in the horse. However, it's highly variable nature and complex inheritance make it difficult for breeders to select homozygous animals for breeding stock in order to increase their production of desirable patterns.
A whole genome scanning panel of microsatellite markers was used to map LP to a 6 cM region on ECA1 (Terry et al. 2004). Prior to the sequencing of the equine genome, two candidate genes Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) and Oculoctaneous Albinism Type II (OCA2) were suggested based on comparative phenotypes in humans and mice (Terry et al. 2004). Both TRPM1 and OCA2 were FISH mapped to ECA1, to the same interval as LP (Bellone et al. 2006a). One SNP in the equine OCA2 gene has been ruled out as the cause for appaloosa spotting (Bellone et al. 2006b).
TRPM1, also known as Melastatin 1 (MLSN1), is a member of the transient receptor potential (TRP) channel family. Channels in the TRP family may permit Ca2+ entry into hyperpolarized cells, producing intracellular responses linked to the phosphatidylinositol and protein kinase C signal transduction pathways (Clapham et al. 2001). TRPs are important in cellular and somatosensory perception (Nilius, 2007). Defects in a light-gated TRP channel results in a loss of phototransduction in Drosophila (reviewed in Kim, 2004). Although the specific function of TRPM1 in melanogenesis has yet to be described, cellular sensation and intercellular signaling is vital for normal melanocyte migration (reviewed in Steingrimsson et al. 2006). In mice and humans, the promoter region of this gene contains four consensus binding sites for a melanocyte transcription factor, MITF (Hunter et al. 1998; Zhiqi et al. 2004). One of these sites, termed an M-box, is unique to melanocytic expression (Hunter et al. 1998). TRPM1 is downregulated in highly metastatic melanoma cells, suggesting that this protein plays an important role in normal melanogenesis (Duncan et al. 1998).
In humans TRPM1 is expressed in several isoforms (Xu et al. 2001: Fang and Setaluri 2000). The long isoform, termed MLSN-L, is thought to be responsible for Ca2+ influx (Xu et al. 2001). It is possible the large relative expression difference that was detected for the long isoform of TRPM1 may interfere with Ca2+ signaling in the melanocytes and thus participate in the biological mechanisms of appaloosa spotting (Bellone et al., 2008).
The specific function of TRPM1 in melanocytes is unknown. It has been described as a tumor suppressor that may regulate the metastatic potential of melanomas, as its expression declines with increased metastatic potential (Duncan et al. 1998; Deeds et al. 2000; Duncan et al. 2001). Treatment of pigmented melanoma cells with a differentiation inducing agent upregulated the long isoform of this gene (Fang and Setaluri, 2000). TRPM1 therefore has potential roles in Ca2+-dependent signaling related to melanocyte proliferation, differentiation, and/or survival.
One potential role of TRPM1 in melanocyte survival is in interaction with the signaling pathway of the cell surface tyrosine kinase receptor, KIT, and its ligand KITLG. Signaling through the KIT receptor is critical for the growth, survival and migration of melanocyte precursors (reviewed by Erikson, 1993). It has been shown that both phospholipase C activation and Ca2+ influx are important in supporting KIT-positive cells (Berger 2006). Stimulation with KIT ligand while blocking Ca2+ influx led to a novel form of cell death that is termed activation enhanced cell death (AECD) (Gommerman and Berger 1998). It is possible that during melanocyte proliferation and differentiation, when KIT positive cells are being stimulated by the ligand in vivo, the absence of TRPM1 expression may result in decreased Ca2+ influx and ultimately result in AECD. Early melanocyte death could explain LP hypopigmentation patterns.
An association of homozygosity for LP and congenital stationary night blindness (CSNB) has been documented (Sandmeyer et al. 2007 and Sandmeyer et al., 2011). CSNB is characterized by a congenital and non-progressive scotopic visual deficit (Witzel et al. 1977, 1978; Rebhun et al. 1984). Affected horses may exhibit apprehension in dimly lit conditions and may be difficult to train and handle in phototopic (light) and scotopic (dark) conditions (Witzel et al. 1977, 1978; Rebhun et al. 1984). Affected animals occasionally manifest a bilateral dorsomedial strabismus (improper eye alignment) and nystagmus (involuntary eye movement) (Rebhun et al. 1984; Sandmeyer et al. 2007). CSNB is diagnosed by an absent b-wave and a depolarizing a-wave in scotopic (dark-adapted) electroretinography (ERG) (FIG. 2). This ERG pattern is known as a “negative ERG” (Witzel et al. 1977). No morphological or ultrastructural abnormalities have been detected in the retinas of horses with CSNB (Witzel et al. 1977; Sandmeyer et al. 2007). A similar “negative ERG” is seen in the Schubert-Bornshein type of human CSNB (Schubert and Bornshein 1952; Witzel et al. 1978). This type of CSNB is thought to be caused by a defective neural transmission within the retinal rod pathway (Witzel et al. 1977, 1978; Sandmeyer et al. 2007). Rod photoreceptors are most sensitive under scotopic conditions. In the dark, these cells exist in a depolarized state. They hyperpolarize in response to light, and signaling occurs through reductions in glutamate release (Stryer 1991). This hyperpolarization is responsible for the a-wave of the electroretinogram. Normally this results in stimulation of a population of bipolar cells, the ON bipolar cells. The glutamate receptor of the ON bipolar cells is a metabotropic glutamate receptor (MGluR6) and this receptor is expressed only in the retinal bipolar cell layer (Nomura et al. 1994; Nakanishi et al. 1998). The MGluR6 receptors sense the reduction in synaptic glutamate and produce a response that depolarizes the ON bipolar cell (Nakanishi et al. 1998). This depolarization is responsible for the b-wave of the electroretinogram. The ERG characteristics of the Schubert-Bornshein type of CSNB are consistent with a failure in depolarization of the ON bipolar cell (Sandmeyer et al. 2007).
Although the exterior of the eye appears normal in CSNB affected horses, these individuals are blind in low light. Despite the use and breeding of horses with leopard complex spotting for hundreds of years, the association of homozygosity with CSNB was not made until recently due to the difficulty in obtaining a definitive diagnosis, which requires an electroretinagram administered by a Veterinary Ophthalmologist. Although CSNB may not affect the function of an affected horse as a show animal the condition does make necessary careful management to avoid injury, both to the horse and to any handler working with an affected horse in dim light. Breeders and clinicians need to be able to test for LP in order to increase the production of desirable spotting patterns and to more easily and effectively diagnose CSNB.
Decreased expression of TRPM1 has been implicated as the cause for both LP and CSNB (Bellone et al. 2008 and WO/2009/105890). Furthermore, this decreased expression in the horse led others to investigate the role of TRPM1 in ON bipolar cell signalling and in human CSNB. Recently TRPM1 has been shown to be the cation channel closed in response to signalling through MGluR6 and the cause of several forms of human CSNB Bellone et al. 2008; Shen et al. 2009; Morgans et al. 2009; Van Genderen et al. 2009; Audo et al. 2009; Nakamura et al. 2010; Li et al. 2009). LP and CSNB have been fine-mapped in the horse to a 173-kb haplotype on ECA1 and Illumina sequencing identified SNPs in the horse for further investigation (Bellone et al. 2010; WO/2009/105890; US patent application Ser. No. 13/292,688).